•Phenol:dichloromethane (2:1, v:v) surpassed other conventional extraction methods.•Ethanol precipitation (−80 °C, 5 min) was the optimal drying conditions.•The developed workflow provided a ...quantitative range of 0.25–1000 nM.•MS parameters were optimized through the central composite design.
Liquid chromatography tandem mass spectrometry has been a widely used technique for quantifying oligonucleotides in biological samples. However, lack of simple and efficient sample cleanup approach remains a challenge. Our study aimed to evaluate the major factors during the sample pretreatment process for developing optimal sample preparation workflow for oligonucleotides. In this study, we have employed a model formed with rat plasma containing a 16 mer oligonucleotide standard in order to comprehensively optimize the sample preparation procedures. These included liquid-liquid extraction (LLE), solid-phase extraction (SPE), protein precipitation (PPT) and LLE combined with SPE. LLE with phenol: dichloromethane (2:1, v:v) was found to be the most efficient sample cleanup procedure with low cost and less toxicity. Followed by the extraction, ethanol precipitation (-80 °C, 5 min) was determined to be the optimal drying conditions. Also, mass spectrometric parameters were tuned to optimal conditions. It was found that the central composite design suite was proved to be highly practical for optimizing MS parameters. Finally, the thoroughly optimized sample preparation workflow was fully validated. The developed assay provided a quantitative range of 0.25–1000 nM, with accuracy and precision were < 7.45% and < 12.20%, respectively. Matrix effect and carryover were also evaluated and no significant effect was observed.
DNA threading the needle: A new method of single‐molecule detection of 5‐hydroxymethylcytosine (5hmC) in DNA has been developed. Selective thiol substitution of 5hmC (giving SMC) in a single‐step, ...bisulfite‐mediated reaction (see scheme) allows the incorporation of a peptide (yellow sphere) or biotin into DNA. Modified 5hmC bases can be readily distinguished at the single‐molecule level using protein nanopore analysis.
Rationale
Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC‐LC/ESI‐MS) has been proved to be useful for the quality control of oligonucleotides. However, ...the lack of separation for some oligonucleotides using HILIC‐LC/MS has proved problematic. This study aimed to improve the resolving ability of HILIC‐LC/MS.
Methods
The study was performed on a Waters UPLC® system coupled to a Waters LCT premier XE ESI‐TOF mass spectrometer using a Zorbax® RRHD HILIC column (2.1 mm × 100 mm, 1.8 μm). Buffer systems contained triethylammonium acetate (TEAA) and acetonitrile. The effects of the concentration of TEAA and the type of organic modifiers on the separation of oligonucleotides were investigated.
Results
The results showed that the optimum concentration of TEAA is 10 mM and acetonitrile is a better organic solvent than methanol. The addition of TEAA in the HILIC mobile phase improved the separation of N from N + A significantly compared to the HILIC method buffered with ammonium acetate. The IP‐HILIC chromatography has demonstrated that the separation of oligonucleotides is sequence dependent. In addition, the IP‐HILIC method produces a much simpler mass spectrum of an oligonucleotide with very efficient desalting.
Conclusions
The HILIC‐LC/MS method with the addition of TEAA at a MS‐compatible concentration has improved the separation of oligonucleotides. The IP‐HILIC‐LC/MS method also produces very simple mass spectra with high desalting efficiency.
Hydrophilic interaction liquid chromatography (HILIC) is here successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of synthetic ...and chemically modified oligonucleotides. Separation was performed on a 2.1
mm
×
100
mm PEEK ZIC
® HILIC column packed with hydrophilic stationary phase with a permanent zwitterionic functional group and a particle size of 3.5
μm with an average pore diameter of 200
Å. A method was developed to separate homogeneous and heterogeneous oligonucleotides as well as methylated oligonucleotides using a quaternary pumping system containing ammonium acetate and water with an acetonitrile gradient. Analyses of oligonucleotides were performed by LC/MS with a detection limit of 2.5
picomole (20
mer) with signal to noise ratio (S/N) of 4.12. The influence of the eluent composition, type of buffer and its concentration, and organic modifier were also evaluated. The HILIC LC/MS method presented in this paper used common, ‘MS friendly’, mobile phases achieving sensitive and selective oligonucleotide analysis.
Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the β-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a ...modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13–24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification.
Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease worldwide, but understanding its pathogenesis is still limited. In this study, plasma untargeted metabolomics of a discovery ...cohort and targeted analysis of a verification cohort were performed by gas chromatograph mass spectrometry (GC/MS). Univariate and multivariate statistical analysis were utilized to reveal differential metabolites, followed by the construction of biomarker panel through random forest (RF) algorithm. The pathways involved in RA were enriched by differential metabolites using Ingenuity Pathway Analysis (IPA) suite. Untargeted metabolomics revealed eighteen significantly altered metabolites in RA. Among these metabolites, a three‐metabolite marker panel consisting of L‐cysteine, citric acid and L‐glutamine was constructed, using random forest algorithm that could predict RA with high accuracy, sensitivity and specificity based on a multivariate exploratory receiver operator characteristic (ROC) curve analysis. The panel was further validated by support vector machine (SVM) and partial least squares discriminant analysis (PLS‐DA) algorithms, and also verified with targeted metabolomics using a verification cohort. Additionally, the dysregulated taurine biosynthesis pathway in RA was revealed by an integrated analysis of metabolomics and transcriptomics. Our findings in this study not only provided a mechanism underlying RA pathogenesis, but also offered alternative therapeutic targets for RA.
The correlation among stationary phases, ion-pairing reagents (IPR) and sequences for ion-pair reversed-phase liquid chromatography mass spectrometry (IP-RP LC-MS) analysis of oligonucleotide (ODN) ...remains unclear. The present study aimed to evaluate such correlation using particle-packed C18 columns in order to search for the optimal combination among them. Five C18 columns packed with core-shell silica, polymer, porous silica and hybrid particles, respectively, were evaluated for the analysis of synthetic and chemically modified ODNs with six different IPRs. Our results showed that silica-based porous particles, compared to other particles, retained ODN the strongest no matter which IPR was used. Meanwhile, among the six IPRs hexylamine (HA) produced the longest retention for all ODNs, regardless of the types of C18 particles. For the separation of ODNs, C18 columns performed similarly under identical LC conditions. However, the separation ability of C18 columns is highly dependent on the type of IPR and ODN sequences. Moreover, the type of particles has little impact on the signals of ODNs for the majority of synthetic sequences, but such impact could be dramatic for chemically modified sequences. On the other hand, both the type of IPR and ODN sequence have a significant effect on MS signals for synthetic and chemically modified ODNs.
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•Separation ability of C18 columns is highly related to the type of ion-pairing reagent and oligonucleotide (ODN) sequences.•Silica-based porous particles among tested columns retained ODNs the strongest regardless of the type of ion-pairing reagent.•Hexylamine among tested alkylamines produced the longest retention for ODNs regardless of the type of C18 particles.
Sample preparation plays a crucial part in plasma metabolomics. In order to obtain an optimal sample extraction method for gas chromatography mass spectrometry (GC-MS)-based plasma metabolomics, five ...different extraction strategies including protein precipitation, liquid-liquid extraction and solid-phase extraction were evaluated systematically for both plasma untargeted- and targeted-metabolomics. The comprehensive evaluation revealed that the all-in-one sample preparation method, MeOH-MTBE-H2O (1:5:1.5, v/v/v), was the optimal extraction method for both untargeted- and targeted-metabolomics. Next, the optimal sample preparation protocol was applied in plasma metabolomics of osteoarthritis (OA). A panel containing cholesterol, lactic acid, stearic acid, alpha-tocopherol and oxalic acid was selected as candidate biomarker to distinguish OA patients from healthy controls (HC) based on the support vector machine (SVM) classification model. The discriminating capability of the candidate biomarker panel was further validated successfully with logistic regression and principal components analysis (PCA) analysis. Therefore, the panel could potentially act as diagnostic biomarker for osteoarthritis.
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•Methanol precipitation provided the most stable signals among five protocols.•Alkalinizing C18 sorbent can dramatically improve the extraction performance.•MeOH-MTBE-H2O(1:5:1.5, v/v/v) is the optimal extraction method among five methods.•A 5-marker panel could potentially act as diagnostic biomarker for osteoarthritis.
Rationale
The mechanism of lipid metabolism disorder in type 2 diabetes (T2DM) remains unclear. This study aimed to reveal the mechanism underlying dysregulated lipid metabolism in T2DM through bile ...acid metabolism.
Methods
A db/db mouse model was employed to investigate the alteration of bile acid profiles in T2DM. Ultrahigh‐performance liquid chromatography with tandem mass spectrometry was used to quantify the detailed bile acid levels in each compartment of enterohepatic circulation. The pathological change of mouse liver was assessed by liver histology and serum biochemical assays. The expression level of bile acid‐related transporters and synthases was measured with Western blot analysis.
Results
The results showed that T2DM can result in severe liver fat accumulation and liver damage. In addition, compared to the control group, in T2DM mice, bile acid synthesis is reduced, while the level of bile acids is increased at the storage sites and the reabsorption sites, but there are subtle gender differences. Further, the ratio of conjugated bile acids in total bile acid in the liver of T2DM mice increased significantly relative to the control group for both female and male mice.
Conclusions
In T2DM, bile acid metabolism is disordered in both male and female mice, which could be the underlying mechanism of dysregulated lipid metabolism in T2DM.
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